The long-term objectives of this research are to gain an understanding of the mechanisms and cellular regulation of actin dynamics. Studies relating to these objectives will be performed in the budding yeast S. cerevisiae. The goal of Specific Aim I is to elucidate the role of an actin-binding protein, profilin, in the reorganization of the actin cytoskeleton. In order to do this, biochemical studies will be performed using purified components. Purified profilin and actin will be used in actin polymerization assays with other proteins known to interact with profilin. In addition, proteins which affect the activity of profilin on actin polymerization will be identified. Experiments will also be performed to learn whether the activity of profilin is regulated in vivo by complexing with other proteins in a spatial and temporal manner. These studies will promote an understanding of the behavior of profilin in different contexts. The goal of Specific Aim II is to determine the molecular events which regulate the activity of profilin. Genetic approaches will be used to obtain this goal. Conditional-lethal profilin alleles will be generated and examined for their affect on the actin cytoskeleton. In addition, the profilin alleles will be used to identify interacting proteins using genetic screens. Proteins identified genetically may be involved in the regulation of profilin activity. The actin cytoskeleton is important for the maintenance of cell morphology and cell motility. The studies outlined in this research proposal will increase understanding of the dynamics of the actin cytoskeleton, biology which applies to all eukaryotic organisms. In addition, the research proposed is relevant to diseases such as cancer. Cancer cells are morphologically distinct from normal cells, are less adhesive, and are highly motile during metastasis. Understanding how the actin cytoskeleton can rapidly assemble and disassemble may eventually lead to therapies that target metastatic cells.